ES2425474T3 - Methods for isolating the crystalline form I of 5-azacitidine - Google Patents

Abstract

A method for isolating a crystalline form of 5-azacitidine comprising the steps of recrystallizing 5-azacitidine in a solvent mixture comprising dimethyl sulfoxide and at least one co-solvent of the group consisting of ethanol, 2-propanol (isopropyl alcohol), acetonitrile and methyl ethyl ketone, cooling said solvent mixture from a selected temperature to allow said 5-azacitidine to dissolve completely to room temperature; and isolate recrystallized 5-azacitidine

Description

Methods for isolating the crystalline form I of 5-azacitidine

Field of the Invention

The invention relates to the isolation of the crystalline polymorphic Form I of 5-azacitidine (also known as 5 azacitidine and 4-amino-1-β-D-ribofuranosyl-S-triazin-2 (1H) -one). 5-azacitidine can be used in the treatment of diseases, including the treatment of myelodysplastic syndromes (MDS).

Background of the invention

Polymorphs exist as two or more crystalline phases that have different arrangements and / or different conformations of the molecule in a crystalline network. When a solvent molecule (s) is contained within the crystalline network, the resulting crystal is called a pseudopolymorph, or solvate. If the solvent molecule (s) found in the crystalline structure is (are) a water molecule (s), then the pseudopolymorph / solvate is called hydrate. Polymorphic and pseudopolymorphic solids have different physical properties, including those due to packaging, and various thermodynamic, spectroscopic, interfacial and mechanical properties (see H. Brittain, Polymorphism in Pharmaceutical Solids, Marcel Dekker, 15 New York, NY, 1999, pages 1 -2). The polymorphic and pseudopolymorphic forms of the drug substance (also known as the "active pharmaceutical principle" (IFA)) administered as such, or formulated as a pharmaceutical product (also known as the final or finished dosage form, or as the pharmaceutical composition ) are well known and may affect, for example, the solubility, stability, ability to flow, fractability and compressibility of pharmacological substances and the safety and efficacy of products

20 pharmacists (see, for example, Knapman, K Modem Drug Discoveries, March 2000: 53).

5-Azacitidine (also known as azacitidine and 4-amino-1-β-D-ribofuranosyl-S-triazin-2 (1H) -one); designation of the Nation Service Center NSC-102816; Registry Number CAS 320-67-2) has undergone tests promoted by NCI for the treatment of myelodysplastic syndromes (SMD). See Komblith et al., J. Clin. Oncol. 20 (10): 2441-2452 (2002) and Silverman et al., J. Clin. Oncol. 20 (10): 2429-2440 (2002). You can define 5-azacitidine by having a

25 formula of C8H12N4O5, a molecular weight of 244.20 and a structure:

In U.S. Patent Application Serial Number 10 / 390,578 (U.S. Patent No. 6,887,855) entitled "Forms of 5-azacytidine" (filed 5-azacitidine forms) filed on March 17, 2003, describe eight different polymorphic and pseudopolymorphic forms of 5-azacitidine (Forms I-VIII), in addition to one form

30 amorphous Each of the Forms I-VIII have characteristic X-ray Diffraction patterns of dust (XRPD) and are easily distinguished from each other using XRPD.

US 3,817,980 describes the synthesis of certain 5-azapyrimidine nucleosides by reacting a 1-O-acyl-1-O-alkyl derivative or a 1-halo derivative of a blocked sugar residue with a silylated 5azacytosine compound in presence of a Lewis acid. The residue containing 5-azacitidine is crystallized from methanol

35 wet.

M.W. Winkley and R.K. Robins, The Journal of Organic Chemistry (1970) 491-495, describe the synthesis of 5-azacytidine by direct glycosylation of 1,3,5-triazines. The material is crystallized from aqueous ethanol.

US 3,891,623 describes the synthesis of citidine and citidine derivatives by reacting a 4trialkylsilyloxyuridine derivative with ammonia or a primary or secondary amine, or with a salt of a primary amine or

40 secondary in the presence of a tertiary amine.

US 6,723,728 describes polymorphic forms of (-) - and (±) -cis-FTC (4-amino-5-fluoro-1- (2- (hydroxymethyl) -1,3oxathiolan-5-yl) -2 (1H) -pyrimidinone). The hydrated crystalline form of (±) -cis-FTC is obtained by dissolving (±) -cis-FTC in water and recrystallizing the FTC.

GB 1 227 691 A describes the isolation of 1-glycosyl-S-azacytosine as a solid in certain reaction mixtures containing methanol.

A. Piscala et al., Nucleid Acid Chemistry (1978) 435-441 describe the isolation of 5-azacitidine as a solid in a reaction mixture. The reaction was carried out in methanol. The solid was then crystallized from water-acetone.

A. Piscala et al., Collection of Czechoslovak Chemical Communications (1964) 2060-2069, describes the isolation of 5azacitidine as a solid in a reaction mixture. The reaction was carried out in methanol. The solid was then crystallized from water-methanol.

Beisler J.A. et al., Journal of Carbohydrates (1977) 281-299 describe the isolation of 5-azacitidine as a solid in certain reaction mixtures. The reactions were carried out in methanol.

Beisler J.A. et al., Journal of Medicinal Chemistry (1978) 204-208 describe the crystallization of 5-azacitidine in ethanolagua.

In U.S. Patent Application Serial Number 10 / 390,578 (U.S. Patent Number 6,877,855) entitled "Forms of 5-azacytidine" (filed 5-azacitidine forms) filed on March 17, it is shown that This prior art procedure for recrystallization of the crude synthesis product does not control the polymorphic forms of 5-azacitidine. Specifically, the prior art recrystallization process produces either Form I substantially free of other forms, or a mixed phase of Form I / II, that is, a solid material in which 5-azacitidine is present in a mixed phase of polymorphic Form I and polymorphic Form II. Thus, the prior art procedures do not allow the Form I to be reliably set as the only form

20 polymorphic in the pharmacological substance. The present invention provides methods that allow recrystallization of 5azacitidine as the polymorphic Form I in a robust and reproducible manner.

Summary of the invention

The present invention provides methods for the robust and reproducible isolation of 5-azacitidine as polymorphic Form I, substantially free of the other forms. The methods involve recrystallizing 5-azacitidine dissolved in a mixture of DMSO / co-solvent and then collecting the resulting crystals. Pharmaceutical compositions comprising Form I of 5-azacitidine together with an excipient, diluent are also described.

or pharmaceutically acceptable vehicle.

Detailed description of the preferred embodiments

Polymorphic form I of 5-azacitidine

30 In U.S. Patent Application Serial Number 10 / 390,578 (U.S. Patent Number 6,877,855) entitled "Forms of 5-azacytidine" (filed 5-azacitidine forms) filed March 17, described Form I of 5-azacitidine. Table 1 provides the angles 2!, Most prominent relative intensity separations for Form I observed using X-ray Powder Diffraction (XRPD) carried out according to the procedure of Example 4:

Relative intensity

39.1 44.1 31.5 27.1 16.0 35.9 37.0 12.4 100.0 28.0 10.8 51.5 25.6 11.5 10.8 13.4

Table 1: Form I of 5-azacitidine - angles 2 !, separations of the most prominent relative intensities (radiation K∀ of Cu)

Isolation of polymorphic Form I of 5-azacitidine by recrystallization

Form I of 5-azacitidine can be isolated reproducibly, substantially free of other forms by recrystallizing dissolved 5-azacitidine and collecting the resulting crystals. Specifically, first 5-azacitidine is completely dissolved in dimethyl sulfoxide (DMSO).

The 5-azacitidine used to form the solution can be synthesized by any method known in the art; An example synthesis scheme is provided in Example 1. Any polymorphic (s) or pseudopolymorphic form (s) of 5-azacitidine, including mixed phases, can be used to form the solution. 5-azacitidine can also be used

10 amorphous to form the solution. It is preferred, although not necessary, that DMSO is preheated to an elevated temperature in order to ensure that 5-azacitidine dissolves completely. Most preferably, dimethyl sulfoxide (DMSO) is preheated to a temperature in the range of about 40 ° C to about 90 ° C.

After the solvation of 5-azacitidine in DMSO, at least one co is added to the 5-azacitidine solution

A solvent selected from the group consisting of ethanol, 2-propanol (isopropyl alcohol), acetonitrile and methyl ethyl ketone. The use of mixtures of two or more of any of the aforementioned co-solvents is also included within the scope of the invention.

It is preferred, although not necessary, that the co-solvents are preheated before mixing with the DMSO, preferably to a temperature below the temperature at which a substantial portion of the co-solvent would be

20 boiling, most preferably, up to about 50 ° C. It is also preferred, although not necessary, that the co-solvent (s) be added gradually to the DMSO.

After mixing, the mixture of DMSO / co-solvent (s) is then equilibrated at different temperatures in order to promote either a slow recrystallization or a rapid recrystallization of Form I of 5-azacitidine, as described below.

Slow recrystallization means that the co-solvent / DMSO solution is allowed to equilibrate at a temperature in the range of about 0 ° C to about 40 ° C, preferably in the range of about 15 ° C to about 30 ° C and, most preferably, at about room temperature. Slow recrystallization of Form I from 5-azacitidine is carried out using ethanol, isopropyl alcohol, methyl ethyl ketone or acetonitrile.

Rapid recrystallization means that the co-solvent solution is allowed to equilibrate at a temperature below 0 ° C,

30 preferably less than about -10 ° C and, most preferably, about -20 ° C. Rapid recrystallization of Form I from 5-azacitidine is carried out with 2-propanol (isopropyl alcohol) or acetonitrile as co-solvent.

Examples of protocols for the recrystallization of Form I according to the methods described herein are provided below in Examples 2 (slow recrystallization with DMSO as solvent.

Main and ethanol, isopropyl alcohol, acetonitrile or methyl ethyl ketone as co-solvent) and 3 (rapid recrystallization with DMSO as the main solvent and isopropyl alcohol or acetonitrile as co-solvent).

After recrystallization, Form I crystals of 5-azacitidine can be isolated from the co-solvent mixture by any suitable method known in the art. Preferably, Form I crystals are isolated using vacuum filtration through a suitable filter medium or by centrifugation.

40 Using the novel methods provided herein, for the first time it is possible to set the Form I of 5-azacitidine as a pharmacological substance in a reproducible and robust manner. In particular, isopropyl alcohol and acetonitrile reliably produce Form I independent of the cooling rate (either slow recrystallization or rapid recrystallization) and are preferred as recrystallization co-solvents to recover Form I. Most preferably, Form I is isolated using isopropyl alcohol as a co-solvent since the

45 isopropyl alcohol has a Class 3 risk classification (solvent with low toxic potential), while acetonitrile has a Class 2 risk classification (solvent to be limited) according to the International Conference of Harmonization's Guidelines for residual Solvents (“Conference Rules International Harmonization for Residual Solvents ”), July 1997. The use of the DMSO / isopropyl alcohol system allows Form I of 5azacitidine to recover reliably for the first time from solvents of low toxic potential without the need to control

50 recrystallization speed. In the most preferred embodiment, Form I of 5-azacitidine can be recovered simply by dissolving 5-azacitidine in DMSO (preferably heated to a temperature in the range of about 40 ° C to about 90 ° C before the addition of 5-azacitidine ), adding isopropyl alcohol, and allowing the solvent mixture to equilibrate at approximately room temperature.

In some embodiments of the invention, Form I of 5-azacitidine can be recovered from a mixture of DMSO / co

Solvent (s) "initiating crystallization with a small amount of Form I of 5-azacitidine either before or during the addition of the co-solvent (s).

By allowing the isolation of a single polymorphic form, one skilled in the art will appreciate that the present invention allows for the first time the production of 5-azacitidine drug substance with uniform and consistent properties between batches, properties that include, but are not limited to same, solubility and dissolution rate. In turn, this allows to provide 5-azacitidine pharmaceutical product (see below) that

5 also has uniform and consistent properties between lots.

Pharmaceutical formulations

For the most efficient administration of the pharmacological substance of the present invention, it is preferred to prepare a pharmaceutical formulation (also known as the "pharmaceutical product" or "pharmaceutical composition"), preferably in unit dosage form, comprising one or more of the polymorphs of 5-azacitidine and one or more

10 pharmaceutically acceptable carriers, diluents or excipients. Most preferably, Form I of 5-azacitidine prepared according to the methods provided herein is used to prepare the pharmaceutical formulation.

Said pharmaceutical formulation may include a solid form of the present invention that is mixed with at least one pharmaceutically acceptable excipient, diluted by an excipient or surrounded by said vehicle which may be in

15 the shape of a capsule, an envelope, tablet, dragee, tablet, paper or other container. When the excipient serves as a diluent, this may be a solid, semi-solid or liquid material that acts as a vehicle, support or medium for the 5-azacitidine polymorph (s). Thus, the formulations may be in the form of tablets, pills, powders, elixirs, suspensions, emulsions, solutions, syrups, capsules (such as, for example, soft and hard gelatin capsules), suppositories, sterile injectable solutions and sterile packaged powders .

Examples of suitable excipients include starches, gum arabic, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose. The formulations may further include lubricating agents such as, for example, talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweeteners; or flavorings. Polyols, buffers and inert fillers can also be used. Examples of polyols include: mannitol,

25 xylitol, sucrose, maltose, glucose, lactose, dextrose and the like. Suitable buffers include phosphate, citrate, tartrate, succinate and the like. Other inert fillers that can be used include those known in the art and are useful in the preparation of various dosage forms. If desired, solid pharmaceutical compositions may include other components such as fillers and / or granulation agents, and the like. The compositions described herein may be formulated to provide rapid, sustained, controlled or controlled release.

30 delayed pharmacological substance after administration to the patient using methods well known in the art.

For reference only, the 5-azacitidine polymorph (s) can be prepared in the form of dosage units for oral administration. The 5-azacitidine polymorph (s) can be mixed with a solid, powdery vehicle such as, for example, lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives or gelatin, as well as with an antifriction agent such as, for example, magnesium stearate, calcium stearate and polyethylene glycol waxes. The mixture is then pressed in the form of tablets or filled into capsules. If tablets, capsules or Pulvules are desired, such tablets, capsules or Pulvules may be coated with a concentrated sugar solution, which may contain gum arabic, gelatin, talc, titanium dioxide or, with an enamel dissolved in the organic solvent or mixture of volatile organic solvents. This coating is

40 can add various dyes in order to differentiate the tablets with different active compounds or with different amounts of the active compound present.

Soft gelatin capsules may be prepared in which the capsules contain a mixture of the 5-azacitidine polymorph (s) and vegetable oil or non-aqueous water miscible materials such as, for example, polyethylene glycol and the like. Hard gelatin capsules may contain granules or powders of the polymorph of 5

Azacitidine with a powdered solid carrier, such as, for example, lactose, sucrose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives or gelatin.

Typically, tablets are prepared for oral use as follows, although other techniques may be used. The solid substances are gently milled to a desired particle size and homogenized and a binder is suspended in a suitable solvent. The 5-azacitidine polymorph (s) and auxiliary agents are mixed with the binder solution. The resulting mixture is wetted to form a uniform suspension. Typically, wetting causes the particles to aggregate slightly, and the resulting mass is gently pressed through a stainless steel sieve having a desired size. The layers of the mixture are then dried in controlled drying units for a predetermined period of time to achieve a desired particle size and consistency. The granules of the mixture

The dried ones are screened gently to separate the dust present. To this mixture, disintegration, antifriction and nonstick agents are added. Finally, the mixture is pressed into tablets using a machine with the appropriate punches and dies to obtain the desired tablet size.

In the event that the above formulations are for use for parenteral administration, said formulation typically comprises sterile aqueous and non-aqueous injection solutions comprising one or more polymorphs of 5-azacitidine for which the preparations are preferably isotonic with the desired recipient blood. These preparations may contain antioxidants, buffers, bacteriostatics and solutes; which make the isotonic formulation with the blood of the desired receptor. Aqueous and non-aqueous suspensions may include suspending agents and thickening agents. The formulations may be present in containers

5 single-dose and multi-dose, for example, in blisters and hermetically sealed vials. Solutions and suspensions for extemporaneous injection can be prepared from sterile powders, granules and tablets of the type described above.

Liquid preparations are prepared for oral administration in the form of solutions, syrups or suspensions, containing the last two, for example, polymorph (s) of 5-azacitidine, sugar and a mixture of ethanol, water, glycerol

10 and propylene glycol. If desired, such liquid preparations contain colorants, flavorings and saccharin. Thickening agents such as carboxymethyl cellulose can also be used.

As such, the pharmaceutical formulations described herein are preferably prepared in a single dose form, each dose containing from about 5 mg to about 200 mg, more usually about 100 mg of the 5-azacitidine polymorph (s) . In liquid form, the single dose

15 contains from about 5 to about 200 mg, more usually about 100 mg of the 5-azacitidine polymorph (s). The term "single dose form" refers to physically discrete units suitable as unit doses for human subjects / patients or other mammals, each unit containing a predetermined amount of 5-azacitidine polymorph calculated to produce the desired therapeutic effect, in association with, preferably, at least one pharmaceutically acceptable carrier, diluent or excipient.

20 Examples

Example 1 (comparative)

Prior art procedure for the synthesis and recrystallization of pharmacological substance 5-azacitidine

5-Azacytidine can be synthesized using 5-azacytosine and 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (RTA) commercially available according to the following route:

The crude synthesis product is dissolved in DMSO (preheated to about 90 ° C), and then methanol is added to the DMSO solution. The co-solvent mixture is equilibrated at approximately -20 ° C to allow the formation of 5-azacitidine crystals. The product is collected by vacuum filtration and allowed to air dry.

Example 2

30 Form I of 5-azacitidine: Slow recrystallization of 5-azacitidine in co-solvent systems

Approximately 250 mg of 5-azacitidine was dissolved with approximately 5 ml of dimethyl sulfoxide (DMSO), preheated to approximately 90 ° C, in separate beakers of 100 ml. The solids were allowed to dissolve to a clear solution. Approximately 45 ml of ethanol, isopropyl alcohol, acetonitrile or methyl ethyl ketone as co-solvent, preheated to about 50 ° C, and the solution were added to the solution.

The resulting was mixed. The solution was covered and allowed to equilibrate under ambient conditions. The product was collected by vacuum filtration using a Buchner funnel.

Example 3

Form I of 5-azacitidine: Rapid recrystallization of 5-azacitidine in co-solvent systems

Approximately 250 mg of 5-azacitidine was dissolved with approximately 5 ml of DMSO, preheated to approximately 90 ° C, in separate beakers of 100 ml. The solids were allowed to dissolve to a clear solution. Approximately 45 ml of isopropyl alcohol or acetonitrile was added to the solution as

5 co-solvent, preheated to about 50 ° C, and the resulting solution was mixed. The solution was covered and placed in a freezer to equilibrate to approximately -20 ° C to allow crystals to form. The solutions were removed from the freezer after crystal formation. The product was collected by vacuum filtration using a Buchner funnel.

Example 4

10 X-ray powder diffraction of recrystallized 5-azacitidine

X-ray powder diffraction patterns (XRPD) were obtained for each sample on a Scintag XDS 2000 or Scintag X2! /! Diffractometer! which operated with 45 kV and 40 mA copper radiation using a Kevex Psi Peltier refrigerated silicon detector or a Thermo ARL Peltier refrigerated solid state detector. To obtain data, 2 or 4 mm source slits and 0.5 or 0.3 mm detector slits were used. The recrystallized material was ground gently

15 for about 1 minute using a biker and an agate hand. The samples were placed in stainless steel or silicon sample holders and leveled using a glass microscope holder. The powder diffraction patterns of the samples were obtained from 2 to 42 ° 2! at 1 ° / minute. The X2 diffractometer calibration was verified using a silicon powder pattern.

The XRPD carried out according to this method revealed that Form I of 5-azacitidine was isolated in Example 2 by

Slow recrystallization using ethanol, isopropyl alcohol, acetonitrile or methyl ethyl ketone as co-solvent and, in Example 3 by rapid recrystallization using isopropyl alcohol or acetonitrile as co-solvent. The results indicate that Form I of 5-azacitidine can be reliably recovered in solvent systems of DMSO / isopropyl alcohol and DMSO / acetonitrile without controlling the rate of recrystallization.

Claims (9)

1. A method for isolating a crystalline form of 5-azacitidine comprising the steps of recrystallizing 5azacitidine in a solvent mixture comprising dimethyl sulfoxide and at least one co-solvent of the group consisting of ethanol, 2-propanol (isopropyl alcohol), acetonitrile and methyl ethyl ketone, cooling said solvent mixture from a 5 temperature selected to allow said 5-azacitidine to dissolve completely to room temperature; Y isolate recrystallized 5-azacitidine. 2. The method according to claim 1, comprising the steps of: (i) preparing a solution comprising 5-azacitidine and dimethyl sulfoxide; (ii) add at least one co-solvent selected from the group consisting of ethanol, 2-propanol, acetonitrile and 10-methyl ethyl ketone to the 5-azacitidine solution and cool the mixture to room temperature; Y (iii) isolate the recrystallized 5-azacitidine. 3. A method for isolating a crystalline form of 5-azacitidine comprising the steps of recrystallizing 5azacitidine in a solvent mixture comprising dimethyl sulfoxide and at least one co-solvent selected from the group consisting of 2-propanol (isopropyl alcohol) and acetonitrile , cooling said solvent mixture from a 15 temperature selected to allow said 5-azacitidine to dissolve completely to -20 ° C; Y isolate recrystallized 5-azacitidine. 4. The method according to claim 3, comprising the steps of: (i) preparing a solution comprising 5-azacitidine and dimethyl sulfoxide;

(ii)

add at least one co-solvent selected from the group consisting of 2-propanol and acetonitrile to the 5-azacitidine solution and cool the mixture to -20 ° C; Y

(iii) isolate the recrystallized 5-azacitidine.

5.

The method according to any of claims 1-4, wherein said co-solvent is 2-propanol.

6.

The method according to any of claims 1-4, wherein said co-solvent is acetonitrile,

7.

The method according to any of claims 1 and 2, wherein said co-solvent is ethanol.

The method according to any of claims 1 and 2, wherein said co-solvent is methyl ethyl ketone.

9. The method according to any of claims 1 to 8, wherein the crystalline form of 5-azacitidine is characterized by peaks observed using X-ray Diffraction of powder at 12,182, 13,024, 14,399, 16,470, 19,049, 20,182, 23,033, 23,872, 27,135 and 29,277 ° 2 !. 10. The method according to any one of claims 1 to 8, wherein the crystalline form of 5-azacitidine is characterized by the following angles 2! and separations d

Separation d (Å)

7,260

6,792

6,146

5,378

4,655

4,396

3,858

3,724

3,284

3,048